In fluorescence microscopy in particular, the signal-to-noise ratio is a critical parameter due to the generally relatively low intensity of the fluorescence signals. This ratio is determined by the number of photons impinging on the detector, as well as by the detection efficiency and the noise produced by the detector. The detection efficiency is given by the quantum efficiency of the detector, i.e., by the probability that a photon impinging on the detector will actually generate a detection signal. If the detector is operated in the “photon counting” mode, i.e., each photon generates its own detection signal, then the signal-to-noise ratio is essentially derived from the Poisson statistic as √n, n denoting the number of detected photons.
When operating a detector in the photon counting mode, the detector's dead time, or delay, is generally problematic. The dead time signifies that time which elapses after a photon is detected, until the detector is again available for detecting a next photon, thus quasi the time that it takes the detector to process an event.
A detector that is recently gaining in popularity is the avalanche photodiode (APD). APDs exhibit the highest detection probability for light having wavelengths of between approximately 200 nm and 1050 nm, so that they are particularly suited for use in the realm of fluorescent light measurements. In addition, APDs offer a high quantum efficiency.
When working with APDs, the dead time is approximately 50 ns, while in the case of photomultipliers it is somewhat less. To ensure that no photons are lost in the photon counting mode and, in addition, to prevent the APDs from being damaged by exposure to too high a level of impinging radiant flux, the radiation impinging on the detector must be kept at low enough levels. For the operation of a fluorescence microscope, this means, for example, that only a very low luminous intensity can be used to excite the sample to be investigated. As a result, a relatively long period of time is needed to record images of a high quality, i.e., to obtain adequate photon statistics. Consequently, rapid biochemical processes in the sample which take place on a time scale that is faster than that of the image recording, are not accessible to conventional fluorescence microscopes.